Diabetes is a term that refers to a collection of diseases resulting in disordered energy metabolism and varying degrees of blood glucose elevations or hyperglycemia. One of the best characterized forms of the disease is that resulting in immunologically mediated destruction of the insulin secreting pancreatic beta cells. This severe form of the disease is termed Insulin-dependent Diabetes (IDD) since it is associated with progressive insulin deficiency and coincident symptoms such as weight loss, glycosuria and polyuria, and increased thirst or polydipsia. Other terms for this form of diabetes are Type 1 Diabetes (cf. Type 2 Diabetes which results from an inherent resistance to insulin action); Ketosis Prone Diabetes because there is abnormal generation of ketone bodies as a result of excessive breakdown of body fats due to the severe insulin deficiency; or Juvenile Diabetes, since virtually all diabetes that appears in childhood and adolescence is of this type (see Atkinson and Maclaren [1994] N. Engl. J. Med. 331:1428-1436).
Diabetes is a major public health problem, especially in Western countries. The incidence rates vary greatly worldwide, from as high as 40 per 100,000 persons in Finland to as low as 1-2 per 100,000 among Japanese, with the US in between. The peak incidence is during the pubertal years associated with increasing bodily demands for insulin associated with muscle growth. The prevalence rates in the US population under age 20 years is 0.25% and it approaches 0.4% over a lifetime, albeit an estimated 10-20% of patients with Non Insulin-dependent Diabetes (NIDD) or Type 2 or Maturity Onset Diabetes also have, in reality, slowly progressive IDD. Thus, it is estimated that there should be at least 1 million Americans affected by IDD.
Diabetes results in progressive damage to the blood vessels of the body, to a degree that depends upon the severity of hyperglycemia and its duration. The incident mortality rate for IDD has been calculated to be 7-fold higher than for age matched non diabetic controls. Whereas the decade long Diabetes Control and Complications Trial (DCCT) concluded in 1994 by the National Institutes of Health in the US showed that meticulous insulin replacement therapy would slow the appearance of damaged arteries, it was not able to prevent this since blood glucose levels were never kept within normal limits. Ocular complications of diabetes are the leading cause of new blindness in persons of 20-74 years of age. The risk of lower extremity amputation is 15-fold higher in those with diabetes, while more than half of the approximately 125,000 persons undergoing lower limb amputation do so as a direct consequence of diabetes. Approximately 40% of persons undergoing renal transplantations have kidney failure because of their diabetes, and the proportion due to diabetes continues to rise each year. Women with diabetes produce newborn infants with a 7% newborn mortality rate, albeit this outcome can be greatly improved with tight glycemic control during the gestation period. Other complications of diabetes include increased heart disease and stroke, loss of nerve cells or neurones innervating the limbs and intestine, impotence and infertility, cataract formation in the lens of the eyes, increased periodontal disease, and predisposition to infectious diseases especially from bacteria and yeast. Of all patients with diabetes, those with IDD have a disproportionate share of these complications because of its severity and usual early age of onset. In the US, the direct health care costs attributable to diabetes in 1994 have been estimated to exceed $120 billion. Thus it is important that the pathogenesis of IDD be understood and strategies be developed to prevent it as a fully expressed clinical disease.
Patients with IDD are unusually prone to other diseases that have become recognized to have autoimmune origins. These diseases include thyroiditis or Hashimoto disease, Graves disease, Addison disease, atrophic gastritis and pernicious anemia, celiac disease and vitiligo (Maclaren, N. K. [1985] Diabetes Care 8(suppl.):34-38). Evidence that IDD itself has an autoimmune nature began with histological studies of patients that succumbed at diagnosis which indicated that the islets were infiltrated with a chronic inflammatory (lymphocytic) infiltrate termed insulitis. This was supported in the early 1970s by reports of islet cell autoantibodies reactive to antigens within the cytoplasm (ICA) (Lendrum et al. [1975] Lancet 1:880-882) or confined to the islet cell surfaces (ICSA) (Maclaren et al. [1975] Lancet 1:977-1000) as detectable by indirect immunofluorescence. Later it was recognized that many patients also develop autoantibodies to insulin (IAA) before their diagnosis (Palmer et al. [1983] Science 222:1337-1339) as well as to insulin receptors (Maron et al. [1983] Nature 303:817-818). Autoantibodies were also reported to an islet cell protein composition of 64,000 M.Wt. in man (Baekkeskov et al. [1982] Nature 298:167-169), in the Biobreeding (BB) rat model (Baekkeskov et al.[1984] Science 224:1348-1350) and in the Non Obese Diabetic (NOD) mouse model (Atkinson and Maclaren [1988] Diabetes 37:1587-1590). 64 kDa antigen has subsequently been reported to be the lower molecular weight isoform of glutaric acid decarboxylase (GAD.sub.65) (Baekkeskov et al. [1990] Nature 347:151-156) (Kauffman et al. [1992] J. Clin. Invest. 283-292). GAD is an enzyme that converts glutamate into the membrane stabilizing neurotransmitter called gamma amino butyric acid or GABA. In addition to autoantibodies to GAD, peripheral blood mononuclear cells were shown to be autoreactive in patients developing IDD (Atkinson and Maclaren et al. [1992] Lancet 339:458-459; and Harrison et al. [1993] Lancet 341:1365-1369). Indeed a leading possible cause for IDD is that immunity to enteroviral proteins (developed through infection by Coxsackie or closely related viruses) that have structural homologies to GAD, may in the genetically predisposed individual, trigger an autoimmune response to islet cells because of this molecular mimicry (Atkinson and Maclaren [1990] Scientific American 262:61-71; Kauffnman et al. [1992] J. Clin. Invest. 89:283-292; Atkinson, Maclaren et al. [1994] J. Clin. Invest. 94:2125-2129).
Since immunological markers predate the clinical onset of IDD often by many years, their possible value in disease prediction became increasingly realized (Maclaren, N. K. [1988] Diabetes 37:1591-1594), permitting in turn options for therapeutically induced delays in diabetes onset to be considered (Muir and Maclaren [1993] J. Autoimmunity 16:301-310). Indeed by 1994, multicenter trials attempting to prevent IDD through prophylactic parenteral insulin or oral insulin therapies had been initiated in the US (the DPT-1 trial), as well as in Europe using prophylactic nicotinamide (the ENDIT trial). Among relatives, the appearance of IAA was shown to predate onset of IDD (Atkinson and Maclaren [1985] Diabetes 35:894-898) while ICA proved to be valuable to the prediction of IDD in relatives (Riley, Maclaren et al. [1990] N. Engl. J. Med. 323:1167-1172) as well as in the general population (Schatz, Maclaren et al. [1994] J. Clin. Invest. 93:2403-2407), as modifiable on the basis of coincident IAA (Krischer, Maclaren et al. [1993] J. Clin. Endo. Metab. 77:743-749). While not ideal, the predictability of IDD based upon the ICA test provided the basis for the DPT-1 and ENDIT trials mentioned above. Furthermore, autoantibodies to the 64 kDa islet cell protein also proved to have utility in IDD prediction (Atkinson, Maclaren et al. [1990] Lancet 335:1357-1360), as eventually realized by the chemical assay for autoantibodies to GAD.sub.65 (Schott, Maclaren et al. [1994] J. Autoimmunity 7:865-872). These studies made it important to resolve the nature of all of the islet cell autoantigens involved in the pathogenesis of IDD (Atkinson and Maclaren [1993] J. Clin. Invest. 92:1608-1616). Whereas ICA, as determined by indirect immunofluorescence of human cryocut pancreatic sections, was likely to represent multiple autoantigens (Genovese et al. [1992] Diabetologia 35:385-388), GAD soon proved to be one of these (Atkinson, Maclaren et al. [1993] J. Clin. Invest. 91:350-356). Insulin, however, was not a component of ICA unless the pancreatic sections were first chemically "fixed" before being used as tissue substrate.
Recently, a 3.6-kb cDNA with a 2,937-bp open reading frame was isolated from a human insulinoma subtraction library (ISL-153) as described by Lan et al. ([1994] DNA and Cell Biology 13:505-514, herein incorporated by reference). The predicted amino acid sequence and in-vitro-translated product of IA-2 cDNA revealed a 979-amino acid protein with a PI value of 7.09 and a molecular mass of 105,847 daltons. The protein sequence is consistent with a signal peptide, an extracellular domain, a transmembrane domain and an intracellular domain. The extracellular domain contains an unusual cysteine-rich region following the signal peptide. The intracellular cytoplasmic domain of IA-2 possesses highly conserved regions similar to the catalytic domains in members of the protein tyrosine phosphatase (PTP) family. Northern blot analyses showed that IA-2 mRNA was expressed in five of five freshly isolated human insulinomas, rat and mouse insulinoma cell lines, and in enriched normal mouse islets. It was also found in normal human brain, pituitary, pancreas, and brain tumor cell lines, but not in a variety of other normal or tumor tissues. Based on the sequence and expression data, it appears that IA-2 is a new member of the receptor-type PTP family that is expressed in islet and brain tissues. The involvement of the molecule in beta cell autoimmunity or IDD was queried but was not disclosed or suggested in that work.
More recently, Payton et al. ([1995] J. Clin. Invest. 96:1506-1511) reported on the relationship of 37 kDa and 40 kDa tryptic fragments of islet antigens in IDD to the IA-2 molecule reported by Lan et al., supra. Payton et al. concluded that the 40 kDa fragment is a tryptic product of the IA-2 molecule but that the 37 kDa molecule, while sharing some antigenic determinants with the 40 kDa molecule, was a fragment of an as yet unidentified molecule.
We report herein, for the first time, the amino acid sequence and nucleotide sequence of both the mouse and the human counterparts of a new IDD associated autoantigen, referred to herein as IA-2.beta.. This antigen, alone or in combination with other IDD associated antigens such as IA-2 and GAD.sub.65, is useful in the prediction (diagnosis), treatment (therapy), and prevention (prophylaxis) of diabetes.